Plasma adenosine is increased in sepsis-induced acute lung injury (ALI). Sustained elevation of adenosine in adenosine deaminase (ADA-/-) mice causes increased permeability lung edema. ADA activity is decreased in rats and humans by cigarette smoke (CS) exposure, a risk< factor of ALI. The fundamental hypothesis is that prolonged exposure to increased adenosine increases lung vascular permeability. The long-range goal is to develop novel therapeutic approaches to prevent/treat lung diseases associated with sustained increased adenosine and CS exposure. The overall objective of this proposal is to elucidate the mechanisms of deleterious effects of prolonged adenosine exposure on lung endothelial (EC) barrier function using in vivo and in vitro approaches. Preliminary studies show that subacute CS exposure elevates lung adenosine and worsens lung edema and that prolonged exposure to elevated adenosine causes lung EC barrier dysfunction via transporter (ENTI)-dependent, not receptor-mediated, mitochondrial oxidative stress and p38 activation. I hypothesize that sustained exposure to elevated adenosine causes EC barrier dysfunction, leading to lung edema, via ENT1-dependent uptake and mitochondrial oxidative stress mediated p38 activation. Specific Aim 1 will determine the extent to which ENT1 and oxidative stress mediate sustained elevated adenosine-induced lung edema in animal models. Specific Aim 2 will identify the mechanism of deleterious effects of sustained exposure to elevated adenosine on barrier function in cultured lung microvascular endothelial cells (LMVEC). I anticipate that sustained exposure to elevated adenosine causes ENT1-mediated disruption of EC barrier function and lung edema and that CS increases adenosine and similarly disrupts EC barrier function. The approach is innovative since it uses ADA deficiency as a model for sustained adenosine elevation and a novel model of CS priming ALI. These studies will elucidate a novel pathway for adenosine effects mediated by intracellular uptake of adenosine. Inhibition of ENT1- facilitated adenosine transport and downstream signaling may likely provide significant and greatly needed new approaches to treat diseases associated with sustained elevated adenosine and CS exposure.